Basophil analysis system and method

ABSTRACT

Provided herein are systems and methods for analyzing blood samples, and more specifically for performing a basophil analysis. In one embodiment, the systems and methods include: (a) staining a blood sample with an exclusive cell membrane permeable fluorescent dye; and then (b) using measurements of light scatter and fluorescence emission to distinguish basophils from other WBC sub-populations. In one embodiment, the systems and methods include performing a basophil cluster analysis of the blood sample, based on the combination of light scatter and fluorescence measurements.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/808,211 filed on Jul. 24, 2015, issued as U.S. Pat. No. 9,810,618,which is continuation of U.S. patent Ser. No. 13/456,744 filed on Apr.26, 2012, issued as U.S. Pat. No. 9,091,625, which claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No.61/482,549, titled, Method For Detecting and Analyzing Basophils, andfiled on May 4, 2011, the entire disclosure of which is incorporated byreference herein.

This application is also related to U.S. patent application Ser. No.13/456,729, filed on Apr. 26, 2012, titled “WHITE BLOOD CELL ANALYSISSYSTEM AND METHOD,” issued as U.S. Pat. No. 9,091,624; and U.S. patentapplication Ser. No. 13/456,738, filed on Apr. 26, 2012, titled“NUCLEATED RED BLOOD CELL ANALYSIS SYSTEM AND METHOD,” issued as U.S.Pat. No. 9,103,759, the entire disclosures of which are hereinincorporated by reference in their entirety.

BACKGROUND

This invention relates to hematology systems and methods. Morespecifically, this invention relates to systems and methods foranalyzing blood samples to identify, classify, and/or quantify basophilsin a sample of blood.

Basophils are a white blood cell (WBC) sub-population, which represent1% or less of the total WBC count for a normal blood sample. Basophilsare the least common sub-population of WBCs. Clinically, basophils areprimarily involved in certain inflammatory and allergic reactions.Basophils discharge immune system mediators, such as histamine,serotonin, and heparin, to assist the flow of blood and to prevent bloodclotting.

Traditionally, basophils are identified and counted manually byexamination of microscope slides containing blood sample smears. Theprecision and accuracy of reviewing slides manually, however, isquestionable because basophils are in such low concentrations relativeto other WBC sub-populations. Furthermore, the need for well-trainedmedical technologists, and their associated cost of labor, makes manualreview of slides even less commercially viable.

Alternative techniques for basophil detection include the use ofantibodies in a flow cytometry analysis system. Numerous antibodies,such as, for example, CD203c, CD63 and FCεR1, were found to be sensitiveand specific to basophil surface antigens. However, the cost of basophilantibody assays, the lengthy sample preparation and measurement process,and the requirement of a certified medical technologist having flowcytometry experience, make flow cytometry assay methods unpopular formost hospitals and laboratories.

Instead, basophils are most commonly reported as “best guess” estimateson automated, five-part differential hematology analyzers. In practice,the development of an accurate and efficient basophil assay has been achallenge because: (1) each blood sample is analyzed in less than aminute, including sample aspiration, sample-reagent interaction andincubation, as well as sample measurement, which is an insufficient timeperiod for basophil identification; (2) basophil events number less than1% in most blood samples; (3) basophils and lymphocytes share similaroptical scattering characteristics, thus increasing the likelihood ofmisidentification; and (4) assays specifically designed for basophilswould greatly increase the complexity and cost of the system.

BRIEF SUMMARY

Provided herein are systems and methods for analyzing blood samples, andmore specifically for performing a basophil analysis. In one embodiment,the systems and methods include: (a) staining a blood sample with anexclusive cell membrane permeable fluorescent dye; and then (b) usingmeasurements of light scatter and fluorescence emission to distinguishbasophils from other WBC sub-populations. In one embodiment, the systemsand methods include performing a basophil cluster analysis of the bloodsample, based on the combination of light scatter and fluorescencemeasurements.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated herein, form part ofthe specification. Together with this written description, the drawingsfurther serve to explain the principles of, and to enable a personskilled in the relevant art(s), to make and use the systems and methodspresented. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIG. 1 is a schematic diagram illustrating a hematology instrument.

FIG. 2A is a cytogram of 90° polarized side scatter vs. intermediateangle scatter for a first sample of blood (SAMPLE 1).

FIG. 2B is a cytogram of axial light loss vs. intermediate angle scatterfor SAMPLE 1.

FIG. 3A is a cytogram of 90° polarized side scatter vs. intermediateangle scatter for a second sample of blood (SAMPLE 2).

FIG. 3B is a cytogram of axial light loss vs. intermediate angle scatterfor SAMPLE 2.

FIG. 4 is a cytogram of fluorescence vs. intermediate angle scatter forSAMPLE 1, showing lymphocytes and basophils.

FIG. 5 is a cytogram of fluorescence vs. intermediate angle scatter forSAMPLE 2, showing lymphocytes and basophils.

FIG. 6A is a cytogram of 90° polarized side scatter vs. intermediateangle scatter for SAMPLE 1.

FIG. 6B is a cytogram of axial light loss vs. intermediate angle scatterfor SAMPLE 1.

FIG. 6C is a cytogram of fluorescence vs. intermediate angle scatter forSAMPLE 1.

FIG. 7A is a cytogram of 90° polarized side scatter vs. intermediateangle scatter for a SAMPLE 2.

FIG. 7B is a cytogram of axial light loss vs. intermediate angle scatterfor SAMPLE 2.

FIG. 7C is a cytogram of fluorescence vs. intermediate angle scatter forSAMPLE 2.

FIG. 8 is a plot showing correlation of basophil percentages analyzed bymanual gating vs. basophil percentages determined by flow cytometry.

FIG. 9 is a plot showing correlation of basophil percentages analyzed byclustering vs. basophil percentages determined by flow cytometry.

DETAILED DESCRIPTION

Provided herein are systems and methods for analyzing blood samples, andmore specifically for performing a basophil analysis to identify,classify, and count basophils in a blood sample. In one embodiment, thesystems and methods generally include: (a) staining a blood sample withan exclusive cell membrane permeable fluorescent dye; and then (b) usingmeasurements of light scatter and fluorescence emission to distinguishbasophils from other white blood cell (WBC) sub-populations. Morespecifically, example embodiments include a hematology analyzer having:an excitation source positioned to excite particles within the bloodsample; a plurality of detectors positioned to measure light scatter andfluorescence emission; and a processor configured to (a) receive themeasurements of light scatter and fluorescence emission and (b) performa basophil cluster analysis of the blood sample, based on the receivedmeasurements. The basophil cluster analysis can include a “coarse”clustering of the received measurements, followed by a “fine” clusteringof the received measurements. The fine clustering can utilize amulti-dimensional, probability distance metric as a determinant tocombine similar clusters.

In one embodiment, the systems and methods include the screening ofnuclei-containing events vs. non-nuclei-containing events by means offluorescence staining and a fluorescence triggering strategy. As such,interference from unlysed red blood cells (RBCs), such aslysis-resistant red blood cells (rstRBCs), and RBC fragments issubstantially eliminated prior to subsequent analysis. In other words,the systems and methods described herein utilize at least onefluorescent dye and a fluorescence triggering system to screen eventscontaining nuclei, to thereby accurately and reliably identify andquantify WBC (and WBC sub-populations). A combination of lightscattering information and fluorescence information is then used tofurther separate WBC sub-populations, and basophils specifically. Thesystems and methods disclosed thereby ensure accurate counting anddifferentiation of basophils.

(1) Use of a Plurality of Optical Channels and at Least One FluorescenceChannel for Analysis.

In one embodiment, the blood sample analysis is conducted by means ofMultiple Angle Polarized Scattering Separation technology (MAPSS), withenhancement from fluorescence information. At least one photodiode, orat least one photomultiplier tube, or both at least one photodiode andat least one photomultiplier tube, are needed to detect light scatteredby each blood cell passing through a flow cell. Two or more photodiodesare used for measuring axial light loss (ALL) signals, which measureabout 0° scatter, and intermediate angle scatter (IAS) signals, whichmeasure low angle (e.g., about 3° to about) 15° scatter. Two or morephotomultiplier tubes are used for detecting 90° polarized side scatter(PSS) signals and 90° depolarized side scatter (DSS) signals. Additionalphotomultiplier tubes are needed for fluorescence (FL1) measurementswithin appropriate wavelength range(s), depending on the choice ofwavelength of the source of light. Each event captured on the systemthus exhibits a plurality of dimensions of information, such as ALL, IAS(one or more channels), PSS, DSS, and fluorescence (one or morechannels). The information from these detection channels is used forfurther analysis of blood cells.

FIG. 1 is a schematic diagram illustrating the illumination anddetection optics of an apparatus suitable for hematology analysis(including flow cytometry). Referring now to FIG. 1, an apparatus 10comprises a source of light 12, a front mirror 14 and a rear mirror 16for beam bending, a beam expander module 18 containing a firstcylindrical lens 20 and a second cylindrical lens 22, a focusing lens24, a fine beam adjuster 26, a flow cell 28, a forward scatter lens 30,a bulls-eye detector 32, a first photomultiplier tube 34, a secondphotomultiplier tube 36, and a third photomultiplier tube 38. Thebulls-eye detector 32 has an inner detector 32 a for 0° light scatterand an outer detector 32 b for 7° light scatter.

In the discussion that follows, the source of light is preferably alaser. In alternative embodiments, a laser is selected that emits lightat a wavelength between about 350 nm to about 700 nm; for example, inone embodiment a laser that emits light at about 488 nm is used. Thesource of light 12 can be a vertically polarized air-cooled CoherentCube laser, commercially available from Coherent, Inc., Santa Clara,Calif. Lasers having wavelengths ranging from 350 nm to 700 nm can beused. Operating conditions for the laser are substantially similar tothose of lasers currently used with “CELL-DYN” automated hematologyanalyzers. However, other sources of light can be used as well; such as,for example, lamps (e.g., mercury, xenon).

Additional details relating to the flow cell, the lenses, the focusinglens, the fine-beam adjust mechanism and the laser focusing lens can befound in U.S. Pat. No. 5,631,165, incorporated herein by reference,particularly at column 41, line 32 through column 43, line 11. Theforward optical path system shown in FIG. 1 includes a sphericalplano-convex lens 30 and a two-element photo-diode detector 32 locatedin the back focal plane of the lens. In this configuration, each pointwithin the two-element photodiode detector 32 maps to a specificcollection angle of light from cells moving through the flow cell 28.The detector 32 can be a bulls-eye detector capable of detecting axiallight loss (ALL) and intermediate angle forward scatter (IAS). U.S. Pat.No. 5,631,165 describes various alternatives to this detector at column43, lines 12-52.

The first photomultiplier tube 34 (PMT1) measures depolarized sidescatter (DSS). The second photomultiplier tube 36 (PMT2) measurespolarized side scatter (PSS), and the third photomultiplier tube 38(PMT3) measures fluorescence emission from about 360 nm to about 750 nm,depending upon the fluorescent dye selected and the source of lightemployed. In one embodiment, PMT3 measures fluorescence emission fromabout 440 nm to about 680 nm, or more specifically from about 500 nm toabout 550 nm. The photomultiplier tube collects fluorescent signals in abroad range of wavelengths in order to increase the strength of thesignal. Side-scatter and fluorescent emissions are directed to thesephotomultiplier tubes by dichroic beam splitters 40 and 42, whichtransmit and reflect efficiently at the required wavelengths to enableefficient detection. U.S. Pat. No. 5,631,165 describes variousadditional details relating to the photomultiplier tubes at column 43,line 53 though column 44, line 4.

Sensitivity is enhanced at photomultiplier tubes 34, 36, and 38, whenmeasuring fluorescence, by using an immersion collection system. Theimmersion collection system is one that optically couples the first lens30 to the flow cell 28 by means of a refractive index matching layer,enabling collection of light over a wide angle. U.S. Pat. No. 5,631,165describes various additional details of this optical system at column44, lines 5-31.

The condenser 44 is an optical lens system with aberration correctionsufficient for diffraction limited imaging used in high resolutionmicroscopy. U.S. Pat. No. 5,631,165 describes various additional detailsof this optical system at column 44, lines 32-60.

The functions of other components shown in FIG. 1, i.e., a slit 46, afield lens 48, and a second slit 50, are described in U.S. Pat. No.5,631,165, at column 44, line 63 through column 45, line 26. Opticalfilters 52 or 56 and a polarizer 52 or 56, which are inserted into thelight paths of the photomultiplier tubes to change the wavelength or thepolarization or both the wavelength and the polarization of the detectedlight, are also described in U.S. Pat. No. 5,631,165, at column 44, line63 through column 45, line 26. Optical filters that are suitable for useherein include band-pass filters and long-pass filters.

The photomultiplier tubes 34, 36, and 38 detect either side-scatter(light scattered in a cone whose axis is approximately perpendicular tothe incident laser beam) or fluorescence (light emitted from the cellsat a different wavelength from that of the incident laser beam).

While select portions of U.S. Pat. No. 5,631,165 are referenced above,U.S. Pat. No. 5,631,165 is incorporated herein by reference in itsentirety.

(2) Use of Fluorescent Dye(s).

WBCs contain a relatively high concentration of DNA in their nuclei.When appropriately designed, a fluorescent dye can be used todifferentiate between different sub-populations of WBCs. For example,lymphocytes and basophils have different fluorescence signatures,despite having similar light scatter signatures. Further, mature RBCs donot contain DNA. Therefore, a fluorescent dye can be selected todifferentiate between populations of blood cells. The purpose of the dyeis to penetrate into live cells easily, bind DNA with high affinity, andemit strong fluorescence with adequate Stokes shift when the dye isexcited by an appropriate source of light. The peak absorption of thedye in the visible band may substantially match the wavelength of thesource of light (within 50 nm of the wavelength of the source of light,more preferably, within 25 nm of the wavelength of the source of light),in order to be properly excite the dye and achieve optimal results.

The fluorescent dye selected is preferably: 1) capable of bindingnucleic acids, 2) capable of penetrating cell membranes of WBCs andnRBCs, 3) excitable at a selected wavelength when subjected to a sourceof light, 4) emits fluorescence upon excitation by the source of light,and 5) is biostable and soluble in a liquid. The dye may be selectedfrom group consisting of: acridine orange, SYBR 11, SYBR Green seriesdye, hexidium iodide, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO21, SYTO RNA Select, SYTO 24, SYTO 25 and any equivalents thereof. Thedye is used to “activate” WBCs and nRBCs, “screen out” unlysed RBCs andfragments of RBCs based on a fluorescence trigger configured in thehematology analyzer, and differentiate between sub-populations of WBCs.The dye is typically present at a concentration of from about 0.1 ng/mLto about 0.1 mg/mL. While various dyes are available, the dye selectedis generally paired with the excitation source of the hematologyanalyzer such that a single exclusive dye is used to stain and excitefluorescence emission in nRBCs and all WBC sub-populations intended tobe identified, quantified, and/or analyzed. As such, a single (i.e.,exclusive) dye can be used to identify, quantify, and analyze basophils,amongst all other WBC subpopulations, at once.

In one embodiment, a fluorescent dye is provided in a reagent, withcombinations of 1) at least one surfactant, 2) at least one buffer, 3)at least one salt, and/or 4) at least antimicrobial agent, in sufficientquantities for carrying out staining and activating up to 1,000×10³cells per microliter. The at least one surfactant, such as “TRITON”X-100 or saponin, is used to destroy the membranes of RBC, and reducethe sizes of fragments of RBCs. The at least one surfactant is typicallypresent at a concentration of from about 0.001% to about 5%. The atleast one antimicrobial agent, such as those from “TRIADINE” or“PROCLIN” families, is used to prevent the contamination of the reagentfrom microbes. The concentration of the at least one antimicrobial agentis sufficient to preserve the reagent for the shelf life required. Theat least one buffer, such as phosphate buffered saline (PBS) or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), is used toadjust the pH of reaction mixture for controlling lysis of RBCs andpreserving WBCs. The at least one buffer is typically present at aconcentration of from about 0.01% to about 3%. The pH typically rangesfrom about 3 to about 12. The at least one salt, such as NaCl or Na₂SO₄,is used to adjust the osmolality to increase the effect of lysing and/oroptimize WBC preservation. The at least one salt may be present at aconcentration of from about 0.01% to about 3%. In certain cases, the atleast one buffer can serve as the at least one salt, or the at least onesalt can serve as the at least one buffer.

In general, lower osmolality, or hypotonicity, is used to accelerate thelysis of RBCs. The osmolality typically ranges from about 20 to about250 mOsm. Lysis of RBCs can be made to occur at a temperature above roomtemperature (e.g., between about 30° C. to about 50° C., such as about40° C.) over a relatively short period of time (e.g., less than about 25seconds, less than about 17 seconds, or even less than about 9 seconds),following mixing of the sample of blood and the reagent at a ratio ofabout one part by volume sample to about 35 parts by volume reagent.

The scatter and fluorescence data for analysis is generally collectedwith a plurality of optical channels and at least one fluorescencechannel, as described above.

(3) Use of a Fluorescence Trigger.

Blood cells emit different magnitudes of fluorescence signals uponexcitation of the fluorescent dye by a source of light. The differencesin magnitude of fluorescence signals arise from the quantity of nucleicacids, namely DNA, inside the cells. The greater the quantity of DNA,the greater the likelihood of higher fluorescence signals. Also,efficacy of penetration of cell membranes, size of the dye, bindingkinetics between the dye and DNA, affinity between the dye and DNA, andother factors, affect the fluorescence signals. Mature RBCs emit minimalfluorescence signals because there is no DNA within mature RBCs. UnlysedRBCs or RBC fragments do not emit fluorescence, although they may emitvery weak auto-fluorescence. Basophils have also shown to have differentfluorescence emission properties than lymphocytes.

As such, the systems and methods presented herein use a fluorescencetrigger for collecting and analyzing WBCs and WBC sub-populations. Inone embodiment, prior to basophil/lymphocyte differentiation, afluorescence trigger is set between signals from RBCs and signals fromWBCs (and nRBCs, when present). The optical and fluorescence informationcollected may then be used to distinguish (or differentiate) WBCsub-populations. For example, two-dimensional cytograms can be used toidentify and distinguish particles.

As used herein, the expression “fluorescence information” means datacollected from a fluorescence channel of a hematology analyzer. As usedherein, the expression “fluorescence channel” means a detection device,such as a photomultiplier tube, set at an appropriate wavelength bandfor measuring the quantity of fluorescence emitted from a sample.

EXAMPLES

FIG. 2A is a cytogram of 90° polarized side scatter (PSS) vs.intermediate angle scatter (IAS) for a first sample of blood (SAMPLE 1)containing approximately 1.6% basophils. FIG. 2B is a cytogram of axiallight loss (ALL) vs. IAS for SAMPLE 1. Basophils 100 were separated fromthe rest of the WBC sub-populations by manual gating. FIG. 3A is acytogram of PSS vs. IAS for a second sample of blood (SAMPLE 2)containing approximately 0.2% basophils. FIG. 3B is a cytogram of ALLvs. IAS for SAMPLE 2. Basophils 100 were separated from the rest of theWBC sub-populations by manual gating. FIGS. 2A, 2B, 3A, and 3Billustrated that basophils can be separated by means of small angle sidescatter (i.e., IAS, 3°-15°) and forward scatter (i.e., ALL, 0°-2°).

FIG. 4 is a cytogram of fluorescence (FL1) vs. IAS for SAMPLE 1. Onlylymphocytes 200, basophils 100, and residue monocytes 300 are shown inthe cytogram. FIG. 5 is a cytogram of FL1 vs. IAS for SAMPLE 2. Onlylymphocytes 200, basophils 100, and residue monocytes 300 are shown inthe cytogram. FIGS. 4 and 5 illustrate that fluorescent dye stainingenables further separation of basophils from WBC sub-populations (e.g.,lymphocytes) in one or more fluorescence dimensions. Such separation isa result of the possible variations in potency of membranes, content ofDNA, and staining efficiency among different WBC sub-populations. Forexample, treatment of WBCs with a WBC reagent containing acridine orange(3 μg/mL), at a temperature of 40° C., for a duration of 25 seconds,results in generally higher FL1 signals for lymphocytes, and lower FL1signals for basophils. Thus, identification and quantification ofbasophils can be further facilitated with such a WBC reagent. In otherwords, basophils can be separated from lymphocytes by means offluorescence emission signals when the WBC reagent causes basophils toemit fluorescence at different magnitudes from that emitted bylymphocytes (or other WBC sub-populations).

Further results are shown with reference to FIGS. 6A-6C and 7A-7C. Forexample, FIGS. 6A, 6B, 6C, show the separation of basophils usingmultiple dimensions (i.e., more than two dimensions (e.g., fivedimensions)) for SAMPLE 1. More specifically, FIG. 6A is a cytogram ofPSS vs. IAS for SAMPLE 1. FIG. 6B is a cytogram of ALL vs. IAS forSAMPLE 1. FIG. 6C is a cytogram of FL1 vs. IAS for SAMPLE 1. Basophils100 were separated from the rest of the WBCs by a clustering analysis.FIGS. 7A, 7B, and 7C show the separation of basophils using multipledimensions (i.e., more than two dimensions (e.g., five dimensions)) forSAMPLE 2. More specifically, FIG. 7A is a cytogram of PSS vs. IAS forSAMPLE 2. FIG. 7B is a cytogram of ALL vs. IAS for SAMPLE 2. FIG. 7C isa cytogram of FL1 vs. IAS for SAMPLE 2. Basophils 100 were separatedfrom the rest of the WBCs by clustering analysis.

A comprehensive study of basophils was also conducted to evaluate thesystems and methods described herein. A total of 56 samples of blood,with the percentage of basophils ranging from 0 to approximately 5%,were measured on a prototype analyzer operated according to the methoddescribed herein. The specimens were analyzed by both manual gating anda clustering analysis algorithm, in order to obtain the concentration ofbasophils, i.e., percentage of basophils (alternatively referred toherein as “% BA”). In operation, the clustering analysis algorithmapplies an initial “coarse” clustering, which results in a large numberof clusters. The algorithm then applies a second “fine” clustering step,utilizing a multi-dimensional, probability distance metric as adeterminant to combine similar clusters. As a result, in this secondstep, the number of cells in a cluster is not used as a determinant,thereby preserving clusters having low concentrations of cells, such asbasophils. All optical and fluorescence dimensions, including ALL, IAS,PSS, DSS and FL1, are utilized to determine the basophil cluster.

Reference values were obtained by measuring the same set of samples withan Accuri® C6 flow cytometer, with a dual antibody basophils panel (CD45and FCεR1). CD45 was measured in the FL3 channel and FCεR1 was measuredin the FL4 channel. The results of this study are shown in FIGS. 8 and9. More specifically, FIG. 8 is a plot showing correlation of basophilpercentages analyzed by manual gating vs. basophil percentagesdetermined by a reference flow cytometer. A total of 56 samples weremeasured and included in the correlation plot. FIG. 9 is a plot showingcorrelation of basophil percentages analyzed by clustering vs. basophilpercentages determined by a reference flow cytometer. A total of 56samples were measured and included in the correlation plot. Correlationswere as follows:Y=0.9375X−0.0435; R ²=0.9151  (see FIG. 8: manual gating vs. reference)Y=1.0721X+0.0348; R ²=0.9144  (see FIG. 9: random clustering vs.reference)

As shown, excellent correlations were achieved between the methoddescribed herein and the reference method (i.e., using the Accuri® C6flow cytometer). The separation between basophils and lymphocytes wasquantitatively evaluated by Bhattacharyya distance as well. The meanBhattacharyya distance between basophils and lymphocytes was 4.2±2.0 forthe 56 samples, indicating overall reliable basophils analysis. Therewere only seven samples (12.5%) showing less optimal separation(Bhattacharyya distance <3.0, but all >2.5).

TABLE 1 compares results from the reference (i.e., using the Accuri® C6flow cytometer), manual gating analysis, clustering analysis, andBhattacharyya distance.

TABLE 1 SAMPLE 1 SAMPLE 2 % BA (Reference) 1.60 0.19 % BA (Manualgating) 1.68 0.25 % BA (Cluster analysis) 1.62 0.22 BA-LY (Bhattacharyyadistance) 4.26 5.27

Satisfactory separation of basophils from lymphocytes was achieved byoptimizing the WBC reagent to generate different optical scatter signalsfor basophils and lymphocytes, and utilizing a fluorescent dye to stainWBCs to emit different levels of fluorescence signals. Accuratequantification of basophils can be achieved by the method describedherein. In addition, the method described herein is fast, simple, andcost effective. Basophils can be identified and quantified in under aminute, typically under 30 seconds, with as few as one WBC reagent. Theformulation of the WBC reagent enables basophils to be observed as aseparate population in a plurality of optical dimensions and in at leastone fluorescence dimension. Further, identification and quantificationof basophils can be carried out simultaneously with the identificationand quantification of all WBC sub-populations, in a single assay.

ADDITIONAL EMBODIMENTS

In one embodiment, the systems and methods include: (a) optimizing a WBCreagent by appropriate selection of surfactant(s), concentration(s) ofsurfactant(s), appropriate selection of WBC stabilizing agent(s),concentration(s) of WBC stabilizing agent(s), final pH of the reactionmixture, and osmolality; (b) including a dye for staining nuclei in theWBC reagent to stain WBCs; and (c) analyzing basophils that appear afterprocessing raw data files using an automatic clustering algorithm. Rawdata files may include events having at least five dimensions ofinformation; namely ALL, IAS, PSS, DSS and FL1, along with time tags andother relevant information. The optimizing component enables thedifferentiation of basophils from lymphocytes in at least one of theoptical dimensions.

In another embodiment, there is provided a hematology analyzer forconducting a basophil analysis on a blood sample that has been dyed witha fluorescent dye, wherein the fluorescent dye is cell membranepermeable and nucleic acid binding. The analyzer comprises an excitationsource positioned to excite particles within the blood sample.

In another embodiment, there is provided a method for detectingbasophils by means of an automated hematology analyzer. The methodincludes the steps of: (a) diluting a sample of whole blood with atleast one white blood cell reagent; (b) incubating the diluted sample ofstep (a) for a sufficient period of time within a selected temperaturerange to lyse red blood cells, preserve white blood cells, and allow atleast one fluorescent dye to stain white blood cells; (c) delivering theincubated mixture of step (b) to a flow cell in a stream; (d) excitingthe sample by means of a source of light as the sample passes throughthe flow cell; (e) collecting a plurality of optical scatter signals andat least one fluorescence emission signal simultaneously; and (f)identifying and quantifying basophils by means of the optical scattersignals and fluorescence emission signals collected in step (e). The atleast one white blood cell reagent may comprise: (a) at least onesurfactant, (b) at least one buffer or at least one salt or at least onebuffer and at least one salt, (c) at least one antimicrobial agent, and(d) at least one fluorescent dye, wherein the formulation of the atleast one white blood cell reagent enables basophils to be observed as aseparate population in a plurality of optical dimensions and in at leastone fluorescence dimension. Basophils may be detected and analyzedduring a white blood cell differential analysis, without using a reagentdesignated solely for detecting and analyzing basophils. The basophilsmay be separated from lymphocytes by means of small angle side scatterand forward scatter. The basophils may be separated from lymphocytes bymeans of fluorescence emission signals when basophils emit fluorescenceat a different magnitude from that emitted by lymphocytes. The basophilsmay be analyzed by means of both optical information and fluorescenceinformation. The basophils may differentiated by means of randomclustering through the use of a plurality of dimensions of opticalinformation and at least one dimension of fluorescence information. Thelight source may have a wavelength of from about 350 nm to about 700 nm.The fluorescence emission may be collected at from about 360 nm to about700 nm, by means of band-pass filters or long-pass filters.Identification and quantification of basophils, and identification andquantification of the remainder of white blood cells, may be carried outsimultaneously in a single assay.

In another embodiment, there is provided a method for detectingbasophils by means of an automated hematology analyzer, the methodcomprising the steps of: (a) diluting a sample of whole blood with atleast one white blood cell reagent; (b) incubating the diluted sample ofstep (a) for a sufficient period of time within a selected temperaturerange to lyse red blood cells, preserve white blood cells, and allow atleast one fluorescent dye to stain white blood cells; (c) delivering theincubated mixture of step (b) to a flow cell in a stream; (d) excitingthe sample by means of a source of light as the sample passes throughthe flow cell; (e) collecting a plurality of optical scatter signals andat least one fluorescence emission signal simultaneously; and (f)identifying and quantifying basophils by means of the optical scattersignals and fluorescence emission signals collected in step (e).

A feature of the systems and methods described herein includesseparating basophils from neutrophils, monocytes and eosinophils bymeans of Multiple Angle Polarized Scatter Separation technology.Basophils and the other three white blood cell sub-populations showsubstantially different signals for the axial light loss channel (ALL),which measures 0° scatter, the intermediate angle scatter channel (IAS),which measures 3°-15° scatter, the 90° polarized side scatter channel(PSS), and the 90° depolarized side scatter channel (DSS). Anotherfeature of the method involves separating basophils from lymphocytes byoptical scattering information (such as, IAS information, ALLinformation, and PSS information) and fluorescence information.

In another embodiment, there is provided a hematology analyzer forconducting a basophil analysis on a blood sample that has been dyed witha fluorescent dye, wherein the fluorescent dye is cell membranepermeable and nucleic acid binding. The analyzer includes an excitationsource positioned to excite particles within the blood sample. Theexcitation source can be a laser. The analyzer further includes aplurality of detectors including: (1) an axial light loss detectorpositioned to measure axial light loss from the excited blood sample,(2) an intermediate angle scatter detector positioned to measureintermediate angle scatter from the excited blood sample, (3) a sidescatter detector positioned to measure 90° side scatter from the excitedblood sample, and (4) a fluorescence detector positioned to measurefluorescence emitted from the excited blood sample. The axial light lossdetector can measure axial light loss at 0° scatter. The intermediateangle scatter detector can measure light angle scatter at about 3° toabout 15°. The plurality of detectors can include one or morephotomultiplier tubes. The analyzer further includes a processorconfigured to: (a) receive the measurements of (1) axial light loss, (2)intermediate angle scatter, (3) 90° side scatter, and (4) fluorescencefrom the plurality of detectors, and (b) perform a basophil clusteranalysis of the blood sample, based on all four measurements. Thebasophil cluster analysis may include a coarse clustering of thereceived measurements. The basophil cluster analysis may further includea fine clustering of the received measurements, utilizing amulti-dimensional, probability distance metric as a determinant tocombine similar clusters.

The side scatter detector may be a polarized side scatter detectorpositioned to measure 90° polarized side scatter from the excited bloodsample. The processor thus may be further configured to distinguishbasophils within the blood sample from lymphocytes within the bloodsample, based on the measurements of (1) axial light loss, (2)intermediate angle scatter, (3) 90° polarized side scatter, and (4)fluorescence. The analyzer may also include a depolarized side scatterdetector positioned to measure 90° depolarized side scatter from theexcited blood sample. The processor may further be configured todistinguish basophils within the blood sample from neutrophils,monocytes, and eosinophils within the blood sample, based on themeasurements of (1) axial light loss, (2) intermediate angle scatter,and (3) 90° side scatter. The processor may be further configured topre-screen the received measurements to remove from consideration anyparticles that do not meet the fluorescence threshold.

The hematology analyzer may further comprise an incubation subsystem fordiluting the blood sample with a reagent. The reagent can include thefluorescent dye and a lysing agent. The reagent may include: (a) atleast one surfactant, (b) at least one buffer or at least one salt, (c)at least one antimicrobial agent, and (d) the fluorescent dye. Theincubation subsystem may be configured to incubate the blood sample withthe reagent for a period of time of less than about 30 seconds. Theincubation subsystem may be configured to incubate the blood sample withthe reagent at a temperature ranging from about 30° C. to about 50° C.The incubation subsystem may be configured to incubate the blood samplewith the reagent at a temperature of about 40° C.

In another embodiment, there is provided a method of performing abasophil analysis with an automated hematology analyzer. The methodcomprises: (a) diluting a sample of whole blood with a reagent, whereinthe reagent includes a red blood cells (RBC) lysing agent and a cellmembrane permeable, nucleic acid binding fluorescent dye; (b) incubatingthe diluted blood sample of step (a) for an incubation period of lessthan about 30 seconds, at a temperature ranging from about 30° C. toabout 50° C.; (c) delivering the incubated sample from step (b) to aflow cell in the hematology analyzer; (d) exciting the incubated samplefrom step (c) with an excitation source as the incubated sampletraverses the flow cell; (e) collecting a plurality of light scattersignals and a fluorescence emission signal from the excited sample; and(f) performing a basophil cluster analysis based on all the signalscollected in step (e). The basophil cluster analysis may include acoarse clustering of the collected signals. The basophil clusteranalysis may further include a fine clustering of the collected signals,utilizing a multi-dimensional, probability distance metric as adeterminant to combine similar clusters. The reagent may include: (a) atleast one surfactant, (b) at least one buffer or at least one salt, (c)at least one antimicrobial agent, and (d) at least one fluorescent dye.The excitation source may have a wavelength of from about 350 nm toabout 700 nm. The excitation source may have a wavelength of about 488nm. The plurality of light scatter signals may include: (1) axial lightloss, (2) intermediate angle scatter, and (3) 90° side scatter. Theplurality of light scatter signals may otherwise include: (1) axiallight loss, (2) intermediate angle scatter, (3) 90° polarized sidescatter, and (4) 90° depolarized side scatter.

In one embodiment, the invention is directed towards one or morecomputer systems capable of carrying out the functionality describedherein. For example, any of the method/analysis steps discussed hereinmay be implemented in a computer system having one or more processors, adata communication infrastructure (e.g., a communications bus,cross-over bar, or network), a display interface, and/or a storage ormemory unit. The storage or memory unit may include computer-readablestorage medium with instructions (e.g., control logic or software) that,when executed, cause the processor(s) to perform one or more of thefunctions described herein. The terms “computer-readable storagemedium,” “computer program medium,” and “computer usable medium” areused to generally refer to media such as a removable storage drive,removable storage units, data transmitted via a communicationsinterface, and/or a hard disk installed in a hard disk drive. Suchcomputer program products provide computer software, instructions,and/or data to a computer system, which also serve to transform thecomputer system from a general purpose computer into a special purposecomputer programmed to perform the particular functions describedherein. Where appropriate, the processor, associated components, andequivalent systems and sub-systems thus serve as examples of “means for”performing select operations and functions. Such “means for” performingselect operations and functions also serve to transform a generalpurpose computer into a special purpose computer programmed to performsaid select operations and functions.

The systems and methods thus provide a fast, simple, low cost method ofdetecting and analyzing basophils on automated hematology analyzers. Theassay can be performed within a minute, including no more than 30seconds for sample incubation and no more than 10 seconds for samplemeasurement. One white blood cell reagent can be used to lyse red bloodcells and differentiate white blood cells, including the least commonsub-population of white blood cells, namely basophils.

CONCLUSION

The foregoing description of the invention has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise form disclosed.Other modifications and variations may be possible in light of the aboveteachings. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,and to thereby enable others skilled in the art to best utilize theinvention in various embodiments and various modifications as are suitedto the particular use contemplated. It is intended that the appendedclaims be construed to include other alternative embodiments of theinvention; including equivalent structures, components, methods, andmeans.

The above Detailed Description refers to the accompanying drawings thatillustrate one or more exemplary embodiments. Other embodiments arepossible. Modifications may be made to the embodiment described withoutdeparting from the spirit and scope of the present invention. Therefore,the Detailed Description is not meant to be limiting. Further, theSummary and Abstract sections may set forth one or more, but not allexemplary embodiments of the present invention as contemplated by theinventor(s), and thus, are not intended to limit the present inventionand the appended claims in any way.

What is claimed is:
 1. A hematology analyzer for conducting a basophilanalysis on a blood sample that contains a plurality of basophils andlymphocytes, the analyzer comprising: an excitation source positioned toexcite particles within the blood sample; a detector unit comprising afluorescence detector positioned to measure fluorescence emitted fromthe excited blood sample; and a processor; a non-transitorycomputer-readable memory medium comprising instructions that cause theprocessor to: excite the blood sample with an excitation source in aflow cell; collect a plurality of fluorescence emission signals from theexcited sample; prior to performing a basophil analysis, excludenuclei-free events and retain nuclei-containing events using only afluorescence trigger that is limited to fluorescence emission signalsand is set to a fluorescence magnitude that is greater than fluorescenceemission signals from RBCs, including RBC fragments, and is less thanfluorescence emission signals from white blood cells (WBCs); and performa basophil cluster analysis on the nuclei-containing events.
 2. Thehematology analyzer of claim 1, wherein the basophil cluster analysisincludes a coarse clustering of the received measurements.
 3. Thehematology analyzer of claim 2, wherein the basophil cluster analysisfurther includes a fine clustering of the nuclei-containing events,utilizing a multi-dimensional, probability distance metric as adeterminant to combine similar clusters.
 4. The hematology analyzer ofclaim 1, wherein the detector unit comprises a side scatter detectorthat is a polarized side scatter detector positioned to measure 90°polarized side scatter from the excited blood sample.
 5. The hematologyanalyzer of claim 4, wherein the detector unit further comprises anaxial light loss detector and an intermediate angle scatter detector andwherein the processor is further configured to distinguish basophilswithin the blood sample from lymphocytes within the blood sample, basedon the measurements of (1) axial light loss by the axial light lossdetector, (2) intermediate angle scatter by the intermediate anglescatter detector, (3) 90° polarized side scatter by the polarized sidescatter detector, and (4) fluorescence by the fluorescence detector. 6.The hematology analyzer of claim 4, further comprising: a depolarizedside scatter detector positioned to measure 90° depolarized side scatterfrom the excited blood sample.
 7. The hematology analyzer of claim 1,wherein the detector unit further comprises an axial light lossdetector, an intermediate angle scatter detector, a side scatterdetector and wherein the processor is further configured to distinguishbasophils within the blood sample from neutrophils, monocytes, andeosinophils within the blood sample, based on the measurements of (1)axial light loss by the intermediate angle scatter detector, (2)intermediate angle scatter by the intermediate angle scatter detector,and (3) 90° side scatter by the side scatter detector.
 8. The hematologyanalyzer of claim 1, wherein the detector unit further comprises anaxial light loss detector, an intermediate angle scatter detector, a 90°polarized side scatter detector and wherein the processor is furtherconfigured to distinguish basophils within the blood sample fromneutrophils, monocytes, and eosinophils within the blood sample, basedon the measurements of (1) axial light loss, (2) intermediate anglescatter, (3) 90° polarized side scatter, and (4) fluorescence.
 9. Thehematology analyzer of claim 1, wherein the detector unit furthercomprises a axial light loss detector that measures axial light loss at0° scatter.
 10. The hematology analyzer of claim 1, wherein the detectorunit further comprises a intermediate angle scatter detector thatmeasures light angle scatter at about 3° to about 15°.
 11. Thehematology analyzer of claim 1, wherein the detector unit comprises oneor more photomultiplier tubes.
 12. The hematology analyzer of claim 1,wherein the excitation source is a laser.
 13. The hematology analyzer ofclaim 1, further comprising: an incubation subsystem for diluting theblood sample with a reagent.
 14. The hematology analyzer of claim 13,wherein the reagent includes (a) at least one surfactant, (b) at leastone buffer or at least one salt, (c) at least one antimicrobial agent,and (d) a fluorescent dye.
 15. The hematology analyzer of claim 13,wherein the incubation subsystem is configured to incubate the bloodsample with the reagent for a period of time of less than 30 seconds.16. The hematology analyzer of claim 13, wherein the incubationsubsystem is configured to incubate the blood sample with the reagent ata temperature ranging from 30° C. to 50° C.
 17. The hematology analyzerof claim 13, wherein the incubation subsystem is configured to incubatethe blood sample with the reagent at a temperature of 40° C.
 18. Thehematology analyzer of claim 1, wherein the processor is configured toperform a multi-dimensional basophil cluster analysis on thenuclei-containing events.
 19. The hematology analyzer of claim 1,wherein the non-transitory computer-readable memory medium furthercomprises instructions to dilute the blood sample with a reagent thatincludes a red blood cell (RBC) lysing agent and a cell membranepermeable, nucleic acid binding fluorescent dye.
 20. The hematologyanalyzer of claim 19, wherein the non-transitory computer-readablememory medium further comprises instructions to incubate the dilutedblood sample for an incubation period of time.
 21. The hematologyanalyzer of claim 20, wherein the non-transitory computer-readablememory medium further comprises instructions to deliver the incubatedsample to a flow cell in the hematology analyzer.